Daryl E. Pritchard

Chromium(VI) Induces p53‐Dependent Apoptosis in Diploid Human Lung and Mouse Dermal Fibroblasts

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory‐tract tissue and DNA and are thought to be human lung carcinogens. In general, Cr(VI) is mutagenic and carcinogenic at doses that also evoke some cell death, and we previously showed that the predominant mode of death is apoptosis. Because p53 has been shown to initiate apoptosis after genotoxic insults, the objective of these experiments was to determine whether p53 is activated in and necessary for apoptosis of normal diploid human lung fibroblasts (HLF cells) after chromium exposure. By using annexin(V) staining and fluorescent microscopy, we found that Cr(VI) caused up to 14% of HLF cells to undergo apoptosis within 24 h after exposure. In addition, by using western blotting, we found that p53 protein levels increased fourfold to sixfold after exposure to sodium chromate. Because the major function of p53 is as a transcription factor, it must be translocated from the cytoplasm to the nucleus after chromate exposure to be active. Immunofluorescence studies using an antibody against p53 showed that, after chromate exposure, p53 was located in the nucleus of the treated HLF cells. The necessity of p53 for chromium‐induced apoptosis was examined in two ways. One approach used dermal fibroblasts from p53 wild‐type, heterozygous, and null mice, and the other approach used HLF cells that were transiently transfected with the human papilloma virus E6 gene, which targets p53 for degradation and creates a functional p53‐null cell. These studies showed that chromium‐induced apoptosis was p53 dependent. Mol. Carcinog. 28:111–118, 2000.

Determination of chromium in human lung fibroblast cells using a large bore-direct injection high-efficiency nebulizer with inductively coupled plasma mass spectrometry

A novel method for the determination of chromium in suspensions of human lung fibroblast cells is described by using a large bore–direct injection high efficiency nebulizer (LB-DIHEN) with micro-scale flow injection analysis and inductively coupled plasma mass spectrometric detection. Chromium(VI)-treated cells were first counted and then suspended in a phosphate buffer saline solution. With the use of the method of standard additions, the relative concentration of Cr in ∼ 100 HLF cells/peak was determined at m/z = 50. Because the cells tend to clump and can yield inhomogeneities in the total number analyzed, Mg was used as an internal standard to compensate for the total cell mass. The level of Cr in HLF cells grown in a medium of 100 μM Na2CrO4 for two hours is on the order of 180 fg Cr/cell after correction for the number of cells in each injection.

Resistance to apoptosis, increased growth potential, and altered gene expression in cells that survived genotoxic hexavalent chromium [Cr(VI)] exposure.

Certain hexavalent chromium [Cr(VI)] compounds are known genotoxic respiratory carcinogens, which induce apoptosis as a predominant mode of cell death. Selection of cells that are resistant to apoptosis may be a factor in tumour progression. We developed sub-populations of telomerase-transfected human fibroblasts (BJ-hTERT) that survived a 99% clonogenically lethal exposure to Cr(VI) (B-5Cr). B-5Cr cells were markedly resistant to apoptosis induced by several agents and exhibited increased clonogenic survival, especially at apoptogenic doses. B-5Cr cells did not exhibit altered cellular uptake of Cr(VI) and retained a normal p53 response to Cr(VI) exposure. We conducted large-scale gene expression analysis at different time-points after a secondary genotoxic Cr(VI) insult in B-5Cr and BJ-hTERT cells using Affymetrix Genechip® human genome arrays. Cr(VI) exposure led to differential regulation of many genes, which affect a diverse set of cellular activities such as transcription, signal transduction, stress response, cell adhesion, DNA repair, apoptosis and cell cycle modulation. We compared Cr(VI)-induced altered gene expression in the B-5Cr cells to that in the parental cells and identified 223, 147 and 204 genes with at least a two-fold difference in expression at 4, 8 and 18 h after exposure, respectively. Cluster analysis by gene function revealed altered expression of genes involved in apoptosis, cell cycle regulation and DNA repair. Our data suggest an alteration in gene expression that may favor cell survival and/or incomplete DNA repair after genotoxic exposure. Selection of cells with altered expression of these genes may constitute the early stages of tumour progression.